E. coli clones, 19A31 and 19F5, containing ply gene produced protein molecules that expressed hemolytic activity. A Western blot revealed cross-reaction to polyclonal type 1 pneumolysin antibody. Proteins from both clones showed the same molecular size of 53 Kd. Large quantities (mg) of purified pneumolysin from these clones will be produced for further chemical and immunological characterization. Molecular manipulation has been performed to induce mutation of the ply gene, such as a mutant gene that produces a truncated protein with elimination of hemolytic activity and retention of the antibody binding properties. We have synthesized oligonucleotides that begin at the 3' at a point 1 base (Glu19) or 21 bases (Asp19) from the cysteine codon at base 1,287. The PCR fragments generated are smaller than the ply gene (1,425 bp). Glu19 is 1,286 bp and Asp19 is 1,265 bp. Cloning is currently being carried out. These mutated pneumolysin molecules will be used as carrier proteins for preparation of pneumococcal PS-protein conjugate vaccines.